Abstract

Staphylococcus aureus (S. aureus) is a Gram-positive pathogen causing a variety of infections in humans and animals. Extensive use of antibiotics has led to the emergence of methicillin-resistant S. aureus (MRSA). As an alternative antibacterial agent against drug-resistant S. aureus, a lytic phage, designated SLPW, was isolated from fecal sewage in a pig farm. The SLPW was morphologically classified under Podoviridae and contains a double-stranded DNA genome. The genome of SLPW was 17,861 bp (29.35% G+C) containing 20 open reading frames and lacked regions encoding lysogeny-related integrase gene and cI repressor gene. Phage SLPW showed a broad host range and high efficiency of plating against various types of S. aureus. One-step growth curve showed a short latency period (10 min) and a long lytic period (120 min). Phage SLPW remained stable under a wide range of temperatures or pH and was almost unaffected in chloroform or ultraviolet light. Further, it efficiently lysed MRSA strains in vitro and in vivo. Intraperitoneal phage administration at 1 h post-infection cured the mice and reduced the bacterial expression of inflammatory cytokines in mice. Specifically, the phage SLPW displayed a wide antibacterial spectrum. It was therapeutically effective against intra-abdominal infection in mice harboring different multilocus sequence typing (MLST) types of S. aureus strains. Therefore, phage SLPW is a potential therapeutic agent against MRSA infections.

One-step growth curve of phage SLPW in S. aureus. Phage SLPW was co-incubated with S. aureus ATCC25923 strain cultured at an MOI of 0.1 for 15 min at 37°C. The mixture was centrifuged to remove non-absorbed phage. The re-suspended pellets were incubated at 37°C and sampled at 10 min intervals over a period of 3 h. Phage titer was measured. Results are shown as means ± SEM from triplicate experiments. The latent period was 10 min: interval between the absorption and the beginning of the initial burst. The burst size was estimated at 95.3 PFU per infected cell, which was the ratio of the final count of liberated phage particles to the initial count of infected bacterial cells.

Bacteriolytic activity of SLPW against S. aureus in vitro. Early exponential cultures of S. aureus(A) ATCC25923 and (B) MS3 strains were co-cultured with SLPW phage at MOIs of 0.01, 1, and 100, respectively. S. aureus cultured with a similar volume of phage diluent was used as a control. Results are shown as means ± SEM from triplicate experiments.

Survival curves of mice infected with S. aureus MS3 strain and treated with phage SLPW. Phage (MOIs of 1) was administered intraperitoneally into mice at 0, 1, or 2 h post-infection and the survival rates were recorded. Data shown are representative of three independent experiments using 10 mice per group, and displayed as mean ± SEM.

Survival curves of mice infected with different S. aureus strains and treated with phage SLPW. Phage (MOIs of 1) was injected intraperitoneally into mice at 1 h post-infection and the survival rates were recorded. Infected mice treated with SM buffer served as control. Data shown are representative of three independent experiments using 10 mice per group, and displayed as mean ± SEM.

S. aureus concentrations in the blood and organs of mice treated with phage SLPW at 1 h post-infection. Six mice were sacrificed from each mouse group at different time points. SM buffer was administered to infected mice, which served as control. Results are shown as means ± SEM from triplicate experiments. The Mann-Whitney U-test was used to compare the phage concentration data. Significant differences (P < 0.01) are indicated by asterisks.

Figure 10 Phage SLPW concentrations in the blood and organs of mice treated with phage SLPW at 1 h post-infection. Six mice were sacrificed from each group at 24 h after the experiment. Phage was injected into uninfected mice, which served as controls. Results are shown as means ± SEM from triplicate experiments. The Mann-Whitney U-test was used to compare the phage concentration data. Significant differences (P < 0.01) are indicated by asterisks.

Levels of the pro-inflammatory cytokines IL-1β, IL-6, and TNF-α in the spleens of mice treated with phage SLPW at 1 h after infection. Six mice were sacrificed from each mouse group at different time points. SM buffer was administered to uninfected control mice. The values of pro-inflammatory cytokines in the control groups were normalized to 1.0. Levels of IL-1β, IL-6, and TNF-α mRNA were normalized to β-actin mRNA levels and were expressed as n-fold increases with respect to the control. Results are shown as means ± SEM from triplicate experiments. The Mann-Whitney U-test was used to compare the cytokine levels between infected groups and treatment groups. Significant differences (P < 0.001) are shown by asterisks.